Parathyroid glands (PTGs) control mineral, hormonal, and skeletal homeostasis by adjusting parathyroid hormone (PTH) secretion in response to changes in serum [Ca2+]. It is well documented that activation of homomeric extracellular calcium-sensing receptor (CaSR) by raising serum [Ca2+] suppresses PTH secretion. However, the mechanisms promoting PTH secretion at hypocalcemic and various hyperparathyroidism (HPT) states due to CaSR-deficiency have not been explored. Our pilot data raise a novel hypothesis of a novel autocrine mechanism by which GABA and GABAB1R regulate G protein signaling of the CaSR to promote PTH secretion. Multi-disciplinary approaches to be performed by two highly complementary teams at University of Pittsburgh and University of California San Francisco will be employed to test this hypothesis through 3 specific aims. Aim 1 will first demonstrate the physiopathological relevance of the functional interaction between the CaSR and GABAB1R in PTGs by studying parathyroid cell (PTC)-specific GABAB1R and/or CaSR knockout mice in the contexts of hypocalcemia and different forms of HPT challenges (i.e., CaSR-deficiency or chronical kidney disease) in vivo and human PTGs excised from patients with primary and secondary HPT. Aim 2 will define the biological actions of Gad1/2 in regulating PTG functions by studying the effects of PTC-specific Gad1 and Gad2 double knockout in conditions of Ca2+ deficiency and various HPT states in mice and assessing Gad1/2 and GABA expression in human PTGs excised from patients with primary and secondary HPT. Aim 3 will delineate molecular mechanisms by which the CaSR/GABAB1R heteromers alter efficacy of G-protein activation of the CaSR and its consequence for PTH secretion and GABA production in cultured parathyroid-derived PTH-C1 cells. Optical (FRET, TIRF, BiFC) and biochemical techniques will be used to test the theory that PTH release and GABA synthesis are controlled through mechanisms involving the allosteric action of GABAB1R on CaSR signaling via receptor heteromerization that inhibits Ca2+-mediated Gq/11 and Gi signal transduction and promote PTH secretion. Successful completion of this project will help to develop new regimens to manage PTH hypo- or hyper-secretion and related endocrine and skeletal diseases and prevent unwanted side-effects of GABAB1R agonists and antagonists prescribed to patients with neurological disorders.